The present invention relates generally to methods for identifying patients who have cardiovascular disease and increased risk of developing atherosclerosis. More particularly, the invention relates to the detection of IgG antibodies to platelet activating factor (PAF) in body fluids of patients. The present inventors have shown that elevated concentrations of antibodies to PAF in body fluids is correlated to borderline hypertension and metabolic syndrome, i.e. early cardiovascular disease, which is connected to increased risk of developing early atherosclerosis.
The morbidity and mortality associated with cardiovascular diseases and atherosclerosis in developed countries is higher than that associated with any other disorder. Hypertension is, together with hyperlipidemia, the most prominent risk factor for atherosclerosis. Individuals with borderline hypertension are an example of early cardiovascular disease in general, with endothelial dysfunction and increased risk of atherosclerotic disease, in apparently healthy individuals. Early atherosclerosis manifests itself in the form of cholesterol depositions in the arterial wall. During recent years, it has been convincingly shown that the atherosclerotic process is a chronic inflammation, characterized by presence of activated T cells and monocytes/macrophages. Many of these macrophages have developed into cholesterol-filled foam cells. The deposition is slow and starts at an early age. Clinical symptoms may take years to manifest themselves and are very serious; they include coronary heart disease and stroke. Generally, the disease process will have begun long before these clinical manifestations appear. There are available a number of genetic analysis screening for patients with pre-deposition for atherosclerosis. But it is desirable to have available a diagnostic technique which provides an early warning of the onset of the deposition. The importance of early detection is stressed by the fact that an effective long-term treatment is possible. The present techniques for diagnosing atherosclerosis depend on measuring cholesterol or triglycerid levels in serum or detection of atheromatous lesions, but by the time of detection, the most effective time for treatment has been passed. U.S. Pat. No. 5,731,208 discloses a screening test for atherosclerosis comprising determining the concentration of p-hydroxyphenylaldehyde-lysine in serum or plasma.
The present inventors have found that elevated concentrations of IgG antibodies to platelet activating factor (PAF) in patients are an indicator of cardiovascular diseases which is often accompanied by early atherosclerosis. More specifically, antibodies to PAF (aPAF) are associated with early vascular disease in the form of both borderline hypertension and the metabolic syndrome, both of which are strong risk factors for later stages of atherosclerosis, which give rise to clinical symptoms.
These results demonstrate that antibodies against PAF represent a novel category of anti-phospholipid antibodies (aPL), which are sensitive to early vascular dysfunction and disease, especially early atherosclerosis and hypertension.
aPL in general, especially against cardiolipin have been shown to predict risk of cardiovascular disease, also in autoimmune diseases like systemic lupus erythematosus (SLE) and our data thus indicate that antibodies against PAF is a novel category of aPL, with a potential as a marker also in other autoimmune conditions in addition to cardiovascular disease and atherosclerosis in general. aPL have been related to both arterial and venous thrombosis, and also to spontaneous abortion. These data indicate that antibodies to PAF were much more strongly associated with spontaneous abortion than aPL, and furthermore, that antibodies to PAF was a novel marker for disease activity in SLE.
Antibodies to PAF are therefore relevant also in these other autoimmune vascular-related diseases.
Also antibodies to PAF-like lipids are relevant in this context, one being lysophosphatidylcholine, where the results indicate a comparable profile as the one obtained by PAF antibodies.
Accordingly, it is a principal object of the present invention to provide a diagnostic method or screening test for early atherosclerosis or cardiovascular changes related to early atherosclerosis. It is yet an other object of the invention to provide a kit for assaying the concentrations of aPAF for diagnosing early atherosclerosis or cardiovascular changes.
xe2x80x9cEarly atherosclerosisxe2x80x9d as used herein refers to the very first stage of atherosclerosis, before the clinical onset of symptoms. xe2x80x9cEarly cardiovascular diseasexe2x80x9d as used herein refers to the first stages of cardiovascular disease, as in borderline hypertension and the metabolic syndrome, when atherosclerosis is yet not easy to detect by other methods and has not given rise to disease.
Platelet activating factor (PAF) is a phospholipid inflammatory mediator that is synthesized by a variety of cells, including monocytes and endothelial cells. During oxidation of LDL, PAF-like lipids are produced. PAF may therefore be of importance in pathological processes in the vascular wall like atherosclerosis and hypertension. In a previous report, the existence of antibodies to PAF (aPAF) were described in individuals with phospholipid antibody syndrome (Barquinero et al., 1994.), but nothing has been reported about possible clinical implications of these antibodies and a putative role in cardiovascular disease.
As mentioned above, we have surprisingly shown that concentration of antibodies to PAF (APAF) is an effective indicator of early cardiovascular disease. We have found that antibodies to this particular antigen develop in patients well before the clinical onset of atherosclerosis.
In our study we found that concentration of APAF was 49.3% higher in borderline hypertension men than in normotensive men the. When defining APAF concentrations above mean concentration of control plus two standard deviations (i.e. 0.144+(2xc3x970. 109)=0.362 OD405) as positive, 15 men out of 73 were positive in the borderline hypertension group whereas only 3 men out of 73 were positive in the normotensive group. Antibodies to PAF as a marker for early atherosclerosis may be combined with additional and alternative markers for early atherosclerosis to improve the accuracy of the diagnosis, such as determining the concentrations of cholesterol, blood lipids or p-hydroxyphenylaldehyde-lysine.
Antibodies against PAF (aPAF) may be determined using any of the methods and techniques conventional in the art for such determination. Conveniently, such a method may comprise immunoassay e.g. ELISA or RIA. The immunoassay will conveniently use an antigen (PAF) in immobilized form, e.g. on microtitre plates, membranes or beads, to isolate the target aPAF. In a sandwich assay, the bound antigen may be labelled using additional soluble antibody, which may be monoclonal or polyclonal and which may either carry a label or, more conveniently, may itself be labelled subsequently by reaction with a secondary antibody carrying a label. Suitable labels include radionucleides, fluorescent substances, and enzymes.
Alternatively, a competitive binding assay may be used. Conveniently, the components needed to perform the immunoassay will be supplied in kit form. Such a kit would comprise:
a) an antigen capable of binding to aPAF and, optionally;
a labelled sample of antigen to aPAF or a fragment thereof;
said antigen (a) in non-immobilised form;
a labelled secondary antibody specific to said antigen (c).
The body fluid on which the determination is performed may be any body fluid in which APAF may be located, but conveniently will be or serum or plasma. In some cases it may be convenient to extract the antibodies, or otherwise treat the sample prior to determination.
The invention will now be described in greater detail by reference to the following non-limiting examples: